Amyotrophic lateral sclerosis (ALS) is a catastrophic degenerative disease of motor neurons that inexorably leads to progressive weakness and death. A major breakthrough in ALS research occurred with the link between familial ALS (fALS) and mutant superoxide dismutase (SOD)1. Surprisingly, the pathogenic basis of this mutant protein is most likely a toxic gain of function rather than enzymatic dysfunction. In this proposal, we hypothesize that a toxic function of mutant SOD1 is the inhibition of vascular endothelial growth factor (VEGF) RNA stabilization through disruption of proper RNA-protein interactions. Recent evidence indicates that VEGF is a critical element for neuroprotection during cellular stress, and that its down-regulation in neural tissues contributes to accelerated motor neuron death. One adaptative pathway that promotes VEGF upregulation early in the response to cellular stress is mRNA stabilization. When cells are exposed to stress induced by hypoxia or cytokine exposure (as with neuroinflammation), for example, VEGF mRNA becomes stabilized due to the presence of adenine- and uridine-rich elements (ARE) in the 3' untranslated region (UTR) of the transcript. This response normally results in enhanced and sustained expression of VEGF. However, we postulate that mutant SOD1 inhibits RNA stabilization and prevents the upregulation of VEGF. The resultant loss of neuroprotection may then accelerate motor neuron death. In our preliminary studies, we have observed a significant diminution of VEGF RNA levels in the spinal cords of SOD1 mutant mice, coupled with in vitro data indicating a marked destabilization of the VEGF transcript when mutant SOD1 is expressed. The broad long-term objective of this proposal, therefore, is to characterize the downstream toxic effects of mutant SOD1 on RNA stabilization of growth factors. We propose two specific aims to test the hypothesis that VEGF RNA stabilization is dysfunctional in our in vitro SOD1 mutant models. First, we will compare the effects of cytokine, hypoxic and oxidative stress on VEGF mRNA stabilization and expression in cells of neural- and glial-origin expressing either wild-type or mutant SOD1. We will also test whether the RNA stabilizer, HuR, can reverse the mutant SOD1-induced destabilization of VEGF RNA. Second, we will characterize the aberrant VEGF 3'UTR ribonucleoprotein complex that we have observed in SOD1 mutant cells by examining protein-protein and VEGF RNA-protein interactions with cellular factors critical to RNA stabilization. The impact of this proposal is its potential for identifying a novel "gain of function" mechanism for the SOD1 mutation in fALS. In the broader picture, characterization of adaptative pathways for the upregulation of VEGF (and other neuroprotective growth factors), such as RNA stabilization, may also have implications in the pathogenesis of sporadic ALS and other neurodegenerative disease. [unreadable] [unreadable]